System for Detection of S-Nitrosoproteins

ABSTRACT

The present invention describes a novel, simplified method for detecting and monitoring the presence of nitrosylated proteins, such as S-nitrosoproteins, in a biological sample using fluorescence detection. The present invention further describes a method which can both quantify and identify the nature of nitrosylated proteins, which method is useful for monitoring both normal and disease states, in the development and screening of potential therapeutic drug species.

CLAIM OF PRIORITY

This application is a continuation of U.S. patent application Ser. No.10/319,457, filed on Dec. 12, 2002, now U.S. Pat. No. ______, whichclaims the benefit of U.S. Provisional Application No. 60/339,268, filedon Dec. 12, 2001. The entire contents of the foregoing are herebyincorporated by reference.

US GOVERNMENT RIGHTS

This invention was made with United States Government support underGrant Nos. 1RO1 HL59337, HL10026 and GM57601-01, awarded by the nationalInstitute of Health. The United States Government has certain rights inthe invention.

BACKGROUND OF THE INVENTION

Nitric oxide (NO) has relatively recently been recognized as abiological messenger that reacts with a variety of sulfhydryl-containingmolecules and enzymes to produce S-nitrosylated compounds. Since NO hasa short half-life under physiological conditions, it generally exists inbiological systems as adducts of amino acids, peptides, and proteins(“NO equivalents”). These NO equivalents are usually biologically activein that they behave as NO donors, and thereby possess uniquepharmacological properties. The various targets for nitrosylationinclude serum albumin, tissue-type plasminogen activator,transcriptional activators, glyceraldehyde-3-phosphate dehydrogenase,human immunodeficiency virus protease, hemoglobin, andprotein-phosphotyrosine phosphatase.

Nitrosylation can alter protein conformation, leading to the activationor inactivation of enzymes or receptor proteins. Like phosphorylation,the nitrosylation reaction behaves like a “chemical switch” that allowscells to transmit stimuli from the plasma membrane to the nucleus in ahighly regulated manner. However, the functions and processes ofnitrosylation are difficult to deconvolute, due to the high number ofclosely-related kinases, and due to the lack of currently availabletechnology to easily and accurately measure the extent or presence ofprotein nitrosylation.

Sulfhydryl groups (—SH, also referred to as “thiol”) are among the mostreactive groups found in protein molecules. S-nitrosoproteins,S-nitrosothiols, and protein S-nitrosylation reactions are terms thatrefer to compounds with linkages through the thiol (—SH) group. Thesetypes of compounds play important roles in cell signaling processes suchas neurotransmission, anion channel regulation, host defense and generegulation. The chemical modification of the —SH group in proteins thushas important regulatory implications and can be used as a tool in thediscovery of novel therapeutics.

Chemical modification of thiol groups occurs physiologically viaoxidation reactions yielding either mixed disulfides or S-nitrosylatedcompounds. Alternatively, modification can occur through persulfide andtrisulfide bond formation. The “S-nitrosylation” of proteins refers tothe transfer of nitric oxide (NO) to sulfhydryl groups on proteins.

By way of example, some cysteine proteases such as caspase-3 andcathepsin K have been demonstrated to be inhibited by NO donors. (SeeWang, Peng et al., Inhibition of Papain by S-Nitrosothiols, J. of Biol.Chem., 275, 2000 pp 20467-20473). Cysteine proteases play importantroles in various biological processes. Elevated proteolytic activity ofcysteine proteases is associated with many disease conditions, such asmuscular dystrophy, inflammation, and rheumatoid arthritis. The activesites of cysteine proteases contain a cysteine sulfhydryl group which ishighly sensitive to oxidation.

Compounds such as S-nitrosoglutathione (GSNO) are relevant biologicalmolecules involved in nitrosylation reactions under physiologicalconditions. These compounds are also known to fluoresce, which wouldtheoretically make their detection facile in samples derived frombiological systems. However, identification of S-nitrosoproteins andmeasurement of their concentration following certain cellular events hasproven to be extraordinarily cumbersome, thus extremely limiting itspotential utility.

In light of the significant physiological implications of NO levels, itwould be useful to have a diagnostic technique that can readily detectlevels of NO or NO equivalents, such as S-nitrosothiols and othernitrosylated NO equivalents, to determine whether levels are normal fornormal physiological conditions, or whether a patient has an existing orpredisposition towards a pathophysiological condition. There is aparticular need for procedures that are affordable and manageable, yetsensitive enough to detect levels of NO, or NO-adducts such asS-nitrosothiols. (See Beckman, J. S. et al., Methods in Nitric OxideResearch, Feelisch and Stainler, Wiley, Chichester, U.K., 1996; U.S.Pat. No. 5,891,735 to Stamler).

Representative of prior art approaches to monitoring of nitrosylation,U.S. Pat. No. 5,459,076 to Stamler et al. (incorporated herein byreference) describes a detection method that requires pretreatment withmercurous ion and a protein-precipitating agent. The samples are thenmonitored by chemiluminescence. This method involves cumbersomepretreatment procedures with a toxic mercury compound and, thus,presents considerable difficulties in application. It would be useful tohave a simple procedure with minimal manipulation and without the use ofadditional chemicals.

The present invention is directed to a practical electrophoresis-basedseparation and identification system for cellular nitrosoproteins. Thedetection system meets a recognized need in the art for monitoring of NOin normal states and in disease states, provides a method foridentifying and quantifying NO in normal and in disease states, andwould facilitate the understanding of these processes for thedevelopment of better therapeutic drug species.

SUMMARY OF THE INVENTION

The present invention is directed towards a method for detecting thepresence of nitrosylated species in biological samples. In a preferredembodiment, the biological samples are comprised of amino acid-basedspecies. Preferably, the nitrosylated species are adducts between NO andamino acids, peptides, or proteins. The atoms forming the adducts withNO include carbon, nitrogen, sulfur, and oxygen. Preferably, the adductis between NO and sulfur groups. More preferably, the adduct is anitrosylated protein. Still more preferably, the protein is anitrosothiol, or an “S-nitrosoprotein.” Still more preferably, thenitrosylated protein is S-nitrosoalbumin.

In an embodiment of the invention, the method for detecting the presenceof nitrosylated species in a biological sample comprises the steps ofcontacting the biological sample with developing reagents, exposing thesample to excitation radiation, and detecting the resultant emittedfluorescence.

In a preferred embodiment, the developing reagents comprise afluorescence-developing agent and a molecular species bearing a reactivemoiety capable of transnitrosylation. Preferably, the reactive moiety isa thiol bearing group. More preferably, the molecular species capable oftransnitrosylation is cysteine.

In yet another embodiment, the developing reagents comprise a saturatedsolution of copper (I) chloride. In still another embodiment, thefluorescent agent is 4,5-diaminofluoroscein. In a preferred embodiment,the developing reagents are added to the biological samplesimultaneously. Alternatively, one or more of the developing reagentsare added to the biological sample sequentially. Alternatively oradditionally, the method of detecting nitrosylated species involvesheating or incubating the biological sample to which thefluorescent-developing agent has been added in the presence of ascorbateand carboxyPTIO.

In another embodiment, the method for detecting nitrosylated species ina biological sample is also capable of quantifying the amount ofnitrosylated species in the sample.

In the present invention, the wavelength of the excitation radiation isabout 488 nm. The preferred fluorescent emission is monitored at awavelength of about 530 nm.

In still another embodiment, the biological sample comprises a mixtureof proteins derived from eukaryotic cells. Preferably, the mixture ofproteins is derived from mammalian cells in the absence of metalchelators.

In another embodiment, the method comprises the additional step oftransferring the nitrosylated species to a solid support materialcapable of binding prior to contacting the sample with the developingreagents. The preferred solid supports include nitrocellulose,polyamides, and other synthetic membranes capable of binding aminoacid-based species.

The present invention further provides a method of detecting thepresence of nitrosylated species in a biological sample comprising oneor more amino acid-based species, which involves separating the aminoacid-based species in the sample, contacting each of these species withdeveloping reagents, exposing the species to excitation radiation, anddetecting the emitted fluorescence.

The nitrosylated species in the separated sample comprise an adductbetween NO and an amino acid-based species. The adduct is formed betweenNO and an atom on the amino acid-based species, including carbon,nitrogen, oxygen and sulfur. The preferred adduct is through a sulfuratom. More preferably, the preferred adduct is between NO and a sulfuratom on a protein. Still more preferably, the nitrosylated protein isS-nitrosoalbumin.

In a preferred embodiment, the developing reagents comprise afluorescence-developing agent and a molecular species bearing a reactivemoiety capable of transnitrosylation. Preferably, the reactive moiety onthe molecular species is a thiol group. More preferably, the molecularspecies is cysteine.

In another embodiment, the developing reagents include a saturatedsolution of copper (I) chloride. More preferably, thefluorescence-developing agent includes 4,5-diaminofluoroscein (DAF-2).Preferably, the developing reagents are added to the biological samplesimultaneously. Alternatively, one or more of the developing reagentsare added to the biological sample sequentially.

In another embodiment, the method is capable of quantifying the amountof each separated nitrosylated species in the biological sample.

The method further comprises the additional step of incubating thebiological sample and the developing reagent to elevated temperature inthe presence of ascorbate and carboxyPTIO. Preferably, the sample isheated to around 37° C.

Preferably, the wavelength of the excitation radiation is about 488 nm.Also, the fluorescent emission is monitored at about 530 nm.

Preferably, the mixture of proteins that are separated prior todetection and quantification are derived from eukaryotic cells. Morepreferably, the mixture of proteins are derived from mammalian cells inthe absence of metal chelators prior to separation and prior todetection and quantification. The separation can be achieved by commonlyused methods that rely on the characteristic physical properties of themolecules, such as the charge, size, molecular weights, polarity, etc.Preferred methods of separation include isoelectric focusing, agarosegel electrophoresis, polyacrylamide gel electrophoresis, HPLC, andpreparative chromatography. Most preferred is the method of separationusing gel electrophoresis.

The invention provides the additional step of determining the chemicalidentity of the individual nitrosylated species in the biologicalsample.

Another aspect of the invention is the capability of providing a kit fordetecting nitrosylated species comprising a fluorescence-developingagent and a molecular species bearing a reactive moiety capable ofnitrosylation, and optionally containing a saturated solution of copper(I) chloride. Preferably, the kit contains 4,5-diaminofluoroscein(DAF-2). Also, preferably, the reactive moiety capable oftransnitrosylation is cysteine.

Preferably, the kit provides the capability of detecting thenitrosylated proteins and identifying the nitrosylated proteins in asample. Also, preferably, the kit provides the capability of quantifyingthe amount of nitrosylated species in a biological sample using the kit.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the fluorescence from 8 nM to 1000 nM of S-nitrosoalbumin(SNOBSA) compared to that of the same concentration of native albumin;

FIG. 2 shows the fluorescence from 8 nM to 1000 nM ofS-nitrosoglutathione (GSNO) compared with that of the same concentrationof glutathione; and

FIG. 3 shows the fluorescence of cytoplasmic (Cyto) and mitochondrial(Mito) proteins obtained from undifferentiated (O) or differentiated (D)neuroblastoma cells expressing wild-type (WT) or mutant (G41D)superoxide dismutase. The arrows indicate proteins with increasedfluorescence in the mitochondrial fraction of cells expressing G41D.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “nitrosoprotein” and similar terms encompass any proteinthat has an —NO group linked through a thiol group, oxygen, carbon, ornitrogen group. S-nitrosoproteins, S-nitrosothiols, and proteinS-nitrosylation reactions are terms that refer to compounds with linkagethrough the thiol (—SH) group. These types of compounds play importantroles in cell signaling processes such as neurotransmission, anionchannel regulation, host defense and gene regulation.

Although a burgeoning number of articles describe a role forS-nitrosoproteins and protein S-nitrosylation reactions in cellsignaling processes such as neurotransmission, anion channel regulation,host defense and gene regulation, the detection of nitrosoproteins hasbeen met with limited success. An important implication is in the FASinduced denitrosylation of Capase-3, which allows lymphocyte apoptosisto proceed. (See Mannick J B, et al. Fas-induced capsasedenitrosylation. Science 1999, 284: 65.) A method for determiningalterations in S-nitrosoprotein concentration following cell signalingevents such as the FAS-ligand binding would provide a mechanism formonitoring the progression of apoptosis. The present invention addressesa need for a method for identifying and quantifying levels ofS-nitrosoproteins, and other NO equivalents, that would be useful inmonitoring the levels of NO in normal and in disease states, forfacilitating diagnoses, and in developing more selective drugs for thetreatment of such disease states.

The present invention, in one embodiment, is directed towards animproved, practical electrophoresis-based separation and identificationmethod for cellular S-nitrosoproteins that allows for the identificationof S-nitrosoproteins in general, in addition to the quantification ofalterations in S-nitrosoprotein concentration following cell signalingevents. The preferable method of detection produces a signal that isdirectly proportional to the concentration of the S-nitrosoprotein.

Isolated S-nitrosoproteins (in the absence of catabolic enzymes) aregenerally quite stable. Protein thiol adducts of NO have relatively longhalf-lives under physiologic conditions as compared to free NO, thusmaking detection of nitrosothiol adducts possible.

For the present invention, the source for nitrosoprotein mixtures aretypically derived from eukaryotic cells. Though S-nitrosoproteins may bepresent in virtually all cells, the cell extracts can be prepared from aspecific cell type or tissue of a mammalian species, such as humanneuroblastoma cells. In accordance with a preferred embodiment of thepresent invention, the complex mixture of proteins is prepared frommammalian cells in the absence of metal chelators.

The present invention is directed to a method of detectingS-nitrosoproteins in a biological sample containing a mixture ofproteins based on the transnitrosylation of nitric oxide to a thiolbearing protein or other macromolecule. Fluorescent output from thereaction of 4,5-diaminofluorescein (DAF-2) and nitric oxide isindicative of their presence. By measuring the fluorescence from thereaction, a quantitative measurement of S-nitrosoproteins can also beobtained.

DAF-2 has been previously used only to measure nitric oxide produced bynitric oxide synthase in situ. In accordance with the present invention,DAF-2 is used for an altogether different purpose: that of identifyingnitric oxide evolved from endogenous S-nitrosoproteins. The detectionmethod in this invention is based on correlating the concentration ofthe S-nitrosoproteins to the amount of cumulative light output from theproduct of nitric oxide and DAF-2 following excitation. The nitric oxide(NO) is likely generated from transnitrosylation of NO⁺ from thenitrosoproteins to cysteine, followed by homolytic breakdown ofS-nitrosocysteine to NO and/or from direct reaction of NO⁺ with DAF-2.

The use of S-nitrosocysteine-copper NO evolution as a measurementtechnique for S-nitrosothiols has been recently reported, (see Fang K,et al., Reductive Assays for S-nitrosothiols: Implications formeasurements in biological systems. Biochem Biophys Res Commun 1998;252:535-540) however, this technique has not previously been used inconjunction with fluorescence detection or with gel electrophoresis. Noprevious technique has been successful in identifying S-nitrosoproteinsby gel electrophoresis. The present invention addresses the need in theart for a practical system of identifying S-nitrosoproteins by gelelectrophoresis. Moreover, the present invention offers the additionalcapabilities of detecting S-nitrosoproteins on membranes followingWestern blotting, on a PAGE gel, by Western blot using ultravioletirradiation before or after reaction with DAF-2, and in solutionfollowing protein isolation. In the case of solutions, a fluorescencedetection system for solutions, as opposed to an inverted microscopysetup, is required.

The method of detecting nitrosoproteins thus comprises the steps ofcontacting the biological sample of mixed proteins with a developingreagent, exposing the sample to an excitation light source and detectingthe emitted fluorescence. In accordance with one embodiment, thedeveloping agent is comprised of 4,5-diaminofluorescein (DAF-2) andL-cysteine (or any other suitable molecule that bears a thiol groupcapable of a transnitrosylation reaction) in a saturated solution ofCuCl. More particularly, the developing reagent comprises 100 mML-cysteine in a saturated solution of CuCl (pH 6) to which about 2.5 toabout 10 μM DAF-2 is added immediately before the developing reagent isplaced in contact with the biological sample. Transnitrosylation tocysteine and the reaction with copper augment the sensitivity ofS-nitrosoproteins to detection by DAF-2. In this embodiment, the amountof S-nitrosoproteins present in the sample is determined based on theintensity of the detected fluorescence relative to a standard curvegenerated from known concentrations of S-nitrosoproteins.

Alternatively, the method in the present invention involves incubatingthe proteins at 37° C. with 10-100 μM DAF-2 in a saturated coppersolution containing ascorbate and carboxyPTIO (1-100 nM), or exposed toUV light after incubation with DAF-2. The mixture of proteins can thenbe separated by native polyacrylamide gel electrophoresis. The gel isthen exposed to a fluorescent light source at an excitation wave lengthof 488 nm and scanned on a fluorimager at an emission wave length ofaround 530 nm. The scanned gel or nitrocellulose is observed for bandsof fluorescence from the reaction of N₂O₃ and DAF-2. In this embodiment,the amount of S-nitrosoproteins present in the sample is determinedbased on the intensity of the detected fluorescence relative to astandard curve generated from known concentrations of S-nitrosoproteins.

The proteins of the biological sample can be separated based on theircharge, molecular weight, size, and/or pH using standard techniquesknown to those skilled in the art, before the biological sample iscontacted with the detection reagent. For example, the proteins can beseparated using chromatographic techniques (such as HPLC) or bypolyacrylamide gel electrophoresis. In one embodiment, the complexmixture of proteins is separated on a native gel, and the proteins aretransferred to a nitrocellulose or other synthetic membrane that iscapable of binding proteins before the proteins are contacted with thedetection reagent. Transferring the proteins to a solid matrix mayenhance the signal generated when the separated proteins aresubsequently contacted with the developing reagent and exposed to anexcitation light source.

The developing reagent of the present invention produces a detectablefluorescent signal in the presence of a nitrosoprotein. The developingreagent can be added directly to the mixture containing the proteins, orthe proteins can first be separated based on their physical propertiesand optionally fractionated before contact with the developing reagent.

When the proteins have been separated by agarose, isoelectric focusingor polyacrylamide gel electrophoresis, the surface of the gel (or thesurface of the membrane if the proteins were subsequently transferredfrom the gel to a solid matrix, such as nitrocellulose) is exposed to athin layer of developing reagent that includes 100 mM L-cysteine in asaturated solution of CuCl, pH 6. DAF-2 (2.5-10 μM) is then addedimmediately before contact with the gel. This gel is placed over anemission wavelength filter (at about 515 nm). The gel or nitrocelluloseis then exposed to a fluorescent light source (excitation wave length490 nm) and scanned by a fluorimeter at an emission wave length 515 nMor photographed with a camera.

The scanned gel or nitrocellulose is observed for bands of fluorescence.Additionally or alternatively, the film in a camera is developed tomeasure cumulative light output from the reaction of NO and DAF-2.

In accordance with one embodiment, the nitrosoproteins identified in thegels (based on the emitted fluorescence) can be further characterized bycutting out or otherwise physically isolating the relevant proteinbands. One skilled in the art will appreciate the technique ofpreparative chromatography and other similar methods that allow for theisolation of individual proteins from a mixture separated bychromatographic techniques. The individual proteins can then be furtheranalyzed by techniques known in the art, such as microsequencing or byaddition of monoclonal antibodies.

This invention represents an improvement on several currently availableprior art techniques. First, in the prior art, DAF-2 has been used onlyto measure nitric oxide produced by nitric oxide synthase in situ. Here,however, DAF-2 is employed in a new and direct way which allowsidentification of nitric oxide evolved from endogenous nitrosylatedproteins. The reaction with copper in the presence of PTIO increases thesensitivity to detection by DAF-2. The technique of the presentinvention is thus more sensitive and capable of detecting micromolarconcentrations, and much simpler than the prior art techniques.

Additionally, this invention is capable of the detection of nitrosylatedproteins in solution following protein isolation procedures likechromatography. A fluorescence detection system for solutions, asopposed to an inverted microscopy setup (used for analysis ofS-nitrosylated proteins on gels) would be required. The invention alsoallows for the isolation of individual proteins by cutting or otherwiseremoving fluorescent bands from gels and sequencing and identifying thenitrosylated proteins in cell lysates as shown in FIG. 3. The inventionmay also be used with 2-dimensional gels for proteomic analysis.

Two specific improvements over the prior art of this technique includethe increased sensitivity for identifying very low concentrations ofendogenous nitrosylated proteins and improved signal-to-noise ratio onthe gels. Increasing the sensitivity and improving the signal-to-noiseratio requires variations in the volumes and concentrations of DAF-2solution, UV light administration, buffer compositions, and timing ofapplication. For instance, the assay sensitivity will be increased ifbuffers are developed in which DAF-2 fluorescent intensity is maximized.Additionally, or alternatively, the resolution in measuring nitrosylatedproteins from other “background” proteins can achieved by varying gelcompositions, thickness, electrophoresis voltages, and buffers. Forinstance, the stability of S-nitrosoproteins is enhanced when run onisoelectric focusing gels as opposed to polyacrylamide gels. Finally, itis anticipated that additional known fluorescent probes such asdihydrorhodamine-derivatives can be used in the context of the presentinvention to increase the specificity and sensitivity of the claimedmethod.

EXAMPLE 1

Various concentrations of S-nitrosylated albumin or unmodified albuminwere incubated with 3 μM DAF-2 for 15 minutes at 37° C. An equal volumeof 100 μM CuCl, 1 μM ascorbate and 100 nM PTIO was then added to eachsample and the sample was incubated for another 15 minutes at 37° C. Thesamples were then loaded onto a 12% nondenaturing, nonreducing gel andrun for 30 minutes at 30 volts. The gel was placed on a MolecularDynamics STORM 860 fluorescent Scanner and exposed to an excitationwavelength of 480 nM. The scanner detects all emissions greater than 520nM. The scanned gel in the figure had been incubated in the presence ofCuCl/cys for 1 hour and 20 minutes at the time of scanning.

FIG. 1 shows fluorescence from 100 μm S-nitrosoalbumin (Lanes 1 and 2)compared with that of the same concentration of native albumin (Lane 3).Of note, native albumin is endogenously S-nitrosylated to a limitedextent.

1. A method for detecting the presence of nitrosylated species in abiological sample comprising one or more amino acid-based species, themethod comprising the steps of: (a) separating the amino acid-basedspecies in the sample; (b) contacting the separated species withdeveloping reagents comprising a fluorescence-developing agent thatproduces a detectable signal in the presence of nitric oxide; (c)contacting the separated species with a molecular species bearing athiol moiety capable of transnitrosylation; (d) exposing the separatedspecies to excitation radiation; and (e) detecting emitted fluorescencefrom the fluorescence-developing agent, wherein the emitted fluorescenceindicates the presence of a nitrosylated species in the sample.
 2. Themethod of claim 1, wherein the nitrosylated species comprises an adductbetween NO and an amino acid-based species, and wherein the adduct isformed between NO and an atom on the amino acid-based species selectedfrom the group consisting of sulfur, oxygen, nitrogen and carbon.
 3. Themethod of claim 2, wherein the adduct comprises a nitrosylated protein.4. The method of claim 1, wherein the fluorescence-developing agentcomprises a dihydrorhodamine derivative.
 5. The method of claim 1,wherein the developing reagents further comprise a saturated solution ofcopper (I) chloride.
 6. The method of claim 1, wherein thefluorescence-developing agent comprises 4,5-diaminofluoroscein (DAF-2).7. The method of claim 1 wherein the method is further capable ofquantitating an amount of nitrosylated species detected in thebiological sample.
 8. The method of claim 1, wherein the methodcomprises the additional step of incubating the separated species towhich has been added the fluorescence-developing agent at an elevatedtemperature, in the presence of ascorbate and2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazolin-1-oxyl 3-oxide(carboxyPTIO).
 9. The method of claim 1, wherein the biological sampleis derived from mammalian cells in the absence of metal chelators. 10.The method of claim 1, wherein the method comprises the additional stepof transferring the separated species to a solid support materialcapable of binding the species, prior to contacting the species with thedeveloping reagents.
 11. The method of claim 1, wherein the nitrosylatedspecies comprises an adduct between NO and an amino acid-based species,and wherein the adduct forms between NO and an atom on the aminoacid-based species selected from the group consisting of sulfur, oxygen,nitrogen and carbon.
 12. The method of claim 11, wherein the adductcomprises a nitrosylated protein.
 13. The method of claim 1, wherein themethod is further capable of quantifying an amount of nitrosylatedspecies detected in the biological sample.
 14. The method of claim 1,wherein the biological sample comprises a mixture of proteins derivedfrom eukaryotic cells.
 15. The method of claim 1, wherein the methodcomprises the additional step of transferring the separated species to asolid support material capable of binding the species, prior tocontacting the species with the developing reagents.
 16. The method ofclaim 1, wherein the separation is achieved by a method selected fromthe group consisting of agarose gel electrophoresis, polyacrylamide gelelectrophoresis, isoelectric focusing, High Performance LiquidChromatography (HPLC), and preparative chromatography.
 17. The method ofclaim 1, wherein the method comprises the additional step of determiningthe chemical identity of one or more individual nitrosylated speciesfrom the biological sample.
 18. A kit for detecting nitrosylated speciesin a biological sample, the kit comprising a fluorescence-developingagent that produces a detectable signal in the presence of nitric oxide,a molecular species bearing a thiol moiety capable of nitrosylation, anda saturated solution of copper (I) chloride.
 19. A method foridentifying nitrosylated species in a biological sample using the kit ofclaim
 18. 20. A method for quantifying the amount of nitrosylatedspecies in a biological sample using the kit of claim
 18. 21. The kit ofclaim 18, wherein the fluorescence-developing agent comprises adihydrorhodamine derivative.
 22. The kit of claim 18, wherein thefluorescence-developing agent comprises 4,5-diaminofluoroscein (DAF-2).